Method of inhibiting cancer metastasis

ABSTRACT

The present invention is aimed at providing an anticancer agent which can inhibit the metastasis of cancer cells when used alone. In addition, the present invention is also aimed at providing an anticancer agent which can inhibit the metastasis of cancer cells while causing a small side effect and very small toxicity even for an extended time of administration. The cancer metastasis-inhibiting anticancer agent according to the present invention is characterized by containing sialic acid, its salt, a polymer of sialic acid or a salt of the polymer as an effective ingredient.

this application is the U.S. national stage entry under 35 USC 371 ofPCT/JP96/00268 filed Feb. 8, 1996.

FIELD OF THE INVENTION

The present invention relates to an anticancer agent for remarkablyinhibiting the metastasis of cancer cells, and is aimed at providing amedicine which can completely cure a cancer by inhibiting the metastasisof the cancer cells.

BACKGROUND TECHNIQUE

In order to treat cancers, there have been recently adopted chemicaltherapy using various anticancer agents, immunotherapy for promoting theproduction of antibodies against cancer cells, surgical therapy forextracting cancer cells, radiotherapy for killing cancer cells byirradiation, etc. However, as these therapies have been developed, thesurgical operations or radiotherapy technically suffer their limits, andcannot effectively inhibit the metastasis of the cancers. On the otherhand, although the chemical therapy directly acts upon the cancer cellswith use of the anticancer agents, many of the anticancer agents thencause harmful side effects even upon normal cells of a host. Therefore,the chemical therapy is not necessarily effective for the metastasis ofthe cancer. Further, no excellent effects against the metastasis of thecancers have not been seen in the case of the immunotherapy which is totreat the cancer. Although the therapeutic effects against originalcancers have been largely enhanced, not a few patients become dead bymetastasized cancers provoked by the metastasis of cancer cells, even ifthe original cancers are completely cured. In order to inhibit themetastasis of the cancers, there has strongly demanded development ofmedicines for inhibiting the metastasis of cancer cells. It isconsidered that the cancer cells metastasize over a wide area throughbeing adhered to cell membranes of tissues such as mesenteries.

Although it is recognized that a medicine for inhibiting the metastasisof cancer cells has an effect to inhibit the metastasis of the cancercells when sizofiran is used in combination with a chemotherapy agent, amedicine has not been commercially available to inhibit the metastasisof cancer cells when used alone. Further, although JP-A 60-190,791, JP-A61-83,125 and JP-A 62-223,124 disclose cancer metastasis-inhibitingactions with sialic acid derivatives, they fail to describe sialic acidor its derivative used as an effective ingredient in the presentinvention or the cancer metastasis-inhibiting action thereof.

PROBLEMS TO BE SOLVED

In view of the above problems, the present invention is aimed atproviding an anticancer agent which can inhibit the metastasis of cancercells when used alone. In addition, the present invention is also aimedat providing an anticancer agent which can inhibit the metastasis ofcancer cells while causing a small side effect and very small toxicityeven for an extended time of administration.

Measure to Solve the Problems

The present invention has been accomplished through screening medicinescapable of inhibiting the metastasis of cancer cells from the naturalsphere, and relates to an anticancer agent having sialic acid and/or itssalt as an effective ingredient and exhibiting a cancermetastasis-inhibiting action.

The present invention also relates to an anticancer agent having apolymer of sialic acid and/or a salt of the polymer as an effectiveingredient and exhibiting a cancer metastasis-inhibiting action.

It is preferable that the polymer of sialic acid is a polymer composedof two to thirteen moles of sialic acid. The reason why the upper limitis set upon a thirteen molecule polymer is that it is possible toeffectively fraction and separate up to the thirteen molecule polymers.It is expected that the sialic acid polymers composed of two to thirteenmolecules of sialic acid and the salts thereof have pharmacologicaleffects similar to those of sialic acid and its salt. As the salts ofsialic acid and its polymers, various pharmaceutically allowable saltsmay be used. As the salts of a monomer of sialic acid, a sodium salt, apotassium salt, a calcium salt, and a magnesium salt may be used. As thesalts of the polymers of sialic acid, sodium salts may be used. "Sialicacid" referred to in the present specification and claims means"N-acetylneuraminic acid".

As pharmaceutical preparations of the present invention, oraladministration preparations such as tablets, capsules, powders, etc.,percutaneous absorption preparations such as suppository, vaginalsuppository, etc, and injection preparations for subcutaneousinjections, intraperitoneal injections, intraveneous injections, etc.may be recited. The oral administration preparations are most preferablefor the purpose of preventing diseases, whereas the injectionpreparations are most preferable for the purpose of emergency.

The oral administration preparations, percutaneous absorptionpreparations and injection preparations may be formulated according toordinary medicine-formulating processes. Formulating examples of an oraladministration preparation and an injection preparation may be recitedas follows.

1) A Formulating Example of the Injection Preparation

After 50 g of sialic acid or its polymer was dissolved into 1000 ml ofdistilled water (free from pyrogen), a pH value of the solution wasadjusted to 7.0 by using a solution of caustic soda. The resultant wasfiltered and sterilized according to ordinary methods. The sterilizedfiltrate was sealingly and aseptically charged into a 20 ml ampule,thereby obtaining an injection preparation.

2) A Formulating Example of the Oral Administration Preparation

A capsulated preparation was produced by charging 280 mg of sialic acidor its polymer having passed a 60-mesh sieve into a No. 3 gelatincapsule.

The dosage varies depending upon the age, the sex, the degree of adisease, etc. of a patient, and cannot be generally specified. Withrespect to sialic acid or a sodium salt of its polymer contained in aninjection preparation, it may be administered at a dosage of 1-2000mg/kg, preferably 10-500 mg/kg per day for an adult, and the number oftimes of administrations is one to six per day. Administration throughintraveneous drip infusion is also an effective measure.

Since sialic acid is a substance which is much contained in terminals ofsaccharides present in surface layers of cells constituting the humanbody and in terminals of saccharides of glycoproteins present in theblood and the body fluid, it is a medicine which causes an extremelysmall adverse effects against the human body.

Sialic acid and its polymers used in the present invention may be any ofchemically synthesized products, enzymatically catalyzed synthesizedproducts obtained by using sialic acid aldolase or cytosinemonophosphate-N-acetylneuraminic acid (CMP-NANA) synthesis enzyme orCMP-NANA transferase, and hydrolyzed products of colominic acid obtainedby decomposing colominic acid with an acid. However, sialic acid and itspolymers are not limited to them.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an adhesion-inhibiting effect of sialic acid upon ratM-cells of cancer cells; and

FIG. 2 shows an interaction of sialic acid upon the adhesion-inhibitingeffect on the rat M-cells of the cancer cells of a LFA-1 antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, sialic acid, a salt of sialic acid,a polymer of sialic acid and/or a salt of the sialic acid polymercontained as an effective ingredient in the anticancer agent inhibitsthe cancer cells from being adhered to the cell membranes such asmesenteries, thereby inhibiting the metastasis of the cancer cells.Sialic acid, the salt of sialic acid, the polymer of sialic acid and thesalt of the sialic acid polymer may be added singly as an effectiveingredient or two or more kinds of them may be used in combination.

EXAMPLES

The present inventors employed, as a model system for the measurement ofthe metastasis-inhibiting action upon the cancer cells, a system inwhich rat ascites hepatoma as extremely malignant cancer cells wereadhered to mesenteries of a rat. They also employed a cell system as asimpler technique. As the former cancer cells, AH66F-cells were used,whereas M-cells were used as the latter mesentery cells.

The present inventors screened various substances present in the naturalsphere by using the above-mentioned measuring system. As a consequence,they recognized that sialic acid and its polymers exhibit activity toinhibit the cancer cells from being adhered to ascites hepatoma and thatconspicuous life-prolonging effect was observed in animal experimentsusing rats suffering AH66F ascites hepatoma. In the following, thepresent invention will be exemplified in more detail with reference toexamples.

Example 1

Mesentery cells (M-cells) originating from a rat was cultivated inside acell-cultivating well plate with a Dulbecco's Modification of Eagle'sMedium (DMEM) containing 5% fetal calf serum (FCS). When the cellsspread over the entire face of the well, the cells were washed with DMEMcontaining no FC3. Further, the cells were cultivated with DMEMcontaining 10 μg/ml or 100 μg/ml of sialic acid at 37° C. After 45minutes passed, the well was washed with DMEM, and rat AH66F cells(4×10.sup.∝ cells/well) and sialic acid at a concentration shown in FIG.1 were added together with DMEM. The mixture was cultivated at 37° C.for one hour. After the cultivation, the mixture was stirred for 30second, and a cultivation supernatant liquid was taken into a sampletube. Into each well was added 200 μl of fresh DMEM, and washing wassimilarly carried out under stirring for 15 seconds. This washingoperation was carried out further twice, and the used wash liquid wascombined with the cultivation supernatant liquid. The resultant wassubjected to centrifugal separation at 12,000 rpm for 5 minutes. After asupernatant liquid was removed, 100 μl of fresh DMEM was added tosuspend the cells. Then, the number of the cells was measured by ahaemacytometer under a microscope (the number of non-adhered cells).

As a control, only cancer cells were cultivated, the same operations asmentioned above were effected, and the number of the cells were measured(the number of cells in control).

The adhesive rate (%) of the cancer cells was calculated according tothe following calculating formula. Results are shown in FIG. 1 in whichthe average adhesive rates at concentrations of sialic acid, etc. overfour wells are given.

    Adhesive rate=[1-(number of non-adhered cells)/(number of cells in control)]×100

As shown in Example 1, 10 μg/ml and 100 μg/ml concentrations of sialicacid inhibited the cancer cells from being adhered to mesentery cells by27% and 31%, respectively.

Example 2

Effect of Inhibiting the Adhesion of Cancer Cells With Sialic Acid, etc.Under Coexistence of Anti-LFA-1 antibody

Example 2 was examined in exactly the same manner as in Example 1 exceptthat sialic acid at a concentration of 0 or 100 μg/ml coexisted with 10μg/ml of anti-LFA-1 antibody in the cultivation to contact the M-cellswith the AH66F cells..

Results are shown in FIG. 2.

Under coexistence of rat anti-LFA-1 antibody (antibody to β-chains ofleukocyte function-associated antigen) well known as inhibiting theadhesive activity of the cancer cells, addition of no sialic acidexhibited 40% inhibition, whereas addition of 100 μg/ml exhibited 7%adhesion inhibition. This shows that the adhesion-inhibiting mechanismof sialic acid upon the cancer cells differs from that of sialic theanti-LFA-1 antibody and that a synergistic effect is attained by boththe substances.

Example 3

In the model tests, it was revealed that sialic acid has the effect ofinhibiting the adhesion of the cancer cells. Accordingly, effects ofsialic acid and its polymer upon rats suffering metastasized cancer(AH66F) were examined.

Life-Prolonging Effect of Sialic Acid, etc. Upon the Cancer-SufferingRats

AH66F cancer cells (1×10⁶ cells) were each peritoneally administered toDonryu male rats (six week age, 100-120 g, each group--6 rats).Immediately thereafter, a solution of sialic acid (dosage: 10 mg/kg and100 mg/kg) or physiological saline solution was peritoneallyadministered in a dosage of 0.5 ml (control group). Then, after sialicacid or physiological saline solution was peritoneally administered over2 days, survival dates of the rats were observed.

The life-prolonging percentage was calculated according to the followingformula. Results are shown in Table 1. Life-prolongingpercentage=[(survival dates of treated group)-(survival dates of controlgroup)/(survival dates of control group]×100

                  TABLE 1                                                         ______________________________________                                        Life-prolonging effect of sialic acid                                         upon rats with AH66F cancer cells                                                                           Life-prolonging                                 Treated group     Survival dates                                                                            percentage                                      ______________________________________                                        Control                7.3 ± 0.3                                                                             --                                          Sialic acid                                                                             10 mg/Kg × 3 days                                                                   11.5 ± 0.4                                                                             58%                                                  100 mg/Kg × 3 days                                                                   13.4 ±  0.4                                                                            84%                                         ______________________________________                                    

As a result, it was confirmed that the control group administered withno sialic acid, etc. exhibited the survival dates of 7.3 ±0.3, the groupadministered with 10 mg/Kg of sialic acid exhibited the life-prolongingeffect by 1.6 times as much as that of the former, and the groupadministered with 100 mg/Kg of sialic acid exhibited the life-prolongingeffect by 1.8 times.

Acute Toxicity Test

LD50 in the case of the intraveneous administration to a Wister-strainrats (male) was determined, according to which the sodium salts ofsialic acid and its trimer (pH 7.0) both revealed not less than 2,000mg/Kg.

What is claimed is:
 1. A method of inhibiting a cancer metastasiscomprising administering to a patient in need thereof a cancermetastasis-inhibiting effective amount of a polymer composed of 2 to 13molecules of N-acetylneuraminic acid.